Mating of the stichotrichous ciliate Oxytricha trifallax induces production of a class of 27 nt small RNAs derived from the parental macronucleus.

Ciliated protozoans possess two types of nuclei; a transcriptionally silent micronucleus, which serves as the germ line nucleus, and a transcriptionally active macronucleus, which serves as the somatic nucleus. The macronucleus is derived from a new diploid micronucleus after mating, with epigenetic information contributed by the parental macronucleus serving to guide the formation of the new macronucleus. In the stichotrichous ciliate Oxytricha trifallax, the macronuclear DNA is highly processed to yield gene-sized nanochromosomes with telomeres at each end. Here we report that soon after mating of Oxytricha trifallax, abundant 27 nt small RNAs are produced that are not present prior to mating. We performed next generation sequencing of Oxytricha small RNAs from vegetative and mating cells. Using sequence comparisons between macronuclear and micronuclear versions of genes, we found that the 27 nt RNA class derives from the parental macronucleus, not the developing macronucleus. These small RNAs are produced equally from both strands of macronuclear nanochromosomes, but in a highly non-uniform distribution along the length of the nanochromosome, and with a particular depletion in the 30 nt telomere-proximal positions. This production of small RNAs from the parental macronucleus during macronuclear development stands in contrast to the mechanism of epigenetic control in the distantly related ciliate Tetrahymena. In that species, 28-29 nt scanRNAs are produced from the micronucleus and these micronuclear-derived RNAs serve as epigenetic controllers of macronuclear development. Unlike the Tetrahymena scanRNAs, the Oxytricha macronuclear-derived 27 mers are not modified by 2'O-methylation at their 3' ends. We propose models for the role of these "27macRNAs" in macronuclear development.

Proposed function of the accumulation of plasma membrane-type Ca2+-ATPase mRNA in resting cysts of the ciliate Sterkiella histriomuscorum.

From an mRNA differential-display analysis of the encystment-excystment cycle of the ciliate Sterkiella histriomuscorum, we have isolated an expressed sequence tag encoding a plasma membrane-type Ca2+-ATPase (PMCA). PMCAs are located either in the plasma membranes or in the membranes of intracellular organelles, and their function is to pump calcium either out of the cell or into the intracellular calcium stores, respectively. The S. histriomuscorum macronuclear PMCA gene (ShPMCA) and its corresponding cDNA were cloned; it is the first member of the Ca2+-ATPase family identified in Sterkiella. The predicted protein of 1,065 amino acids exhibits 37% identity with PMCAs of diverse organisms. A phylogenetic analysis showed its relatedness to homologs of two alveolates: the ciliate Paramecium tetraurelia and the apicomplexan Toxoplasma gondii. Overexpression of the protein ShPMCA failed to rescue the wild-type phenotype of three Ca2+-ATPase-defective mutant strains of Saccharomyces cerevisiae; this failure contrasts with the reported ability of the PMCAs of parasites to complement defects in yeast. ShPMCA mRNA is markedly accumulated during encystment and in resting cysts, suggesting a function during excystment. To address the possibility of a signaling role for calcium at excystment, the capacity of calcium to induce excystment was examined.

Not-adaptive behavior of isotropically heated, inert populations of Oxytricha bifaria (Ciliophora, Stichotrichia).

The physiological effects on isotropically heated populations of Oxytricha bifaria cultured at 24 degrees C were investigated. At 34.6 degrees C ciliates became inert, and did not adaptively react to either cold or warm microgradients; they neither moved towards the favorable cold thermal source nor escaped from the unfavorable warm one. The inert oxytrichas were only able to perform the Side-Stepping Reaction (SSR) on the same spot. However, mobile ciliates at 31.6 degrees C reacted to the cold microgradient by immediately orienting themselves towards its source, without accelerating but reducing their SSR frequency. Moreover, in a warm microgradient such ciliates immediately increased their SSR frequency, then moved away from the thermal source. At 34.6 degrees C the behavior of ciliates was not-adaptive--not acting to guide the organisms to more favorable conditions--whereas at 31.6 degrees C it was still clearly adaptive. Therefore, the locomotory inertness of the oxytrichas at 34.6 degrees C was the result of thermal stress rather than their behavioral response to the environmental isotropy, in contrast to populations of the same species made inert at 9 degrees C.

The behavior of the doublet of Oxytricha bifaria (Ciliata, Hypotrichida): a contribution to the understanding of this enigmatic form.

The tracks of normal organisms of Oxytricha bifaria and of stage IA, IB, II, III, IV and V doublets were studied to test the hypothesis that the doublet might function as a dispersal form. Stage IA, the only stage to swim, swims straight with only rare interruptions; its rate of mobility (Rmo = 443 micro/s) is roughly twice that of singlets (Rmo = 218 micro/s). Stage IA doublets swim in three-dimensional movement which enables them to be carried away by water currents. The other stages seem to represent passage back towards the normal singlet form. The ethological evidence reported here together with other results already published supports the working hypothesis that the doublet of O. bifaria is a dispersal form suggests that the doublet might well represent a special fourth differentiation state of this species in addition to pairs, giants, and cysts.

Cold microgradients elicit adaptive behavior in isotropically cooled, inert populations of Oxytricha bifaria (Ciliophora, Hypotrichida).

To complete our investigations on the oriented behavioral response of isotropically cooled, inert populations of Oxytricha bifaria to a warm thermal gradient, their physiological potentialities under cold microgradient conditions arising at 8.5 degrees C were studied. We monitored the behavior of the experimental populations, both at the level of the passing cold wave front, and afterwards when the thermal gradient stabilized, evaluating (i) their distribution in general, (ii) their relative centroids, (iii) the percentage of both backward creeping and immobile ciliates, and (iv) the numerical indices and rates of their creeping tracks. At the arrival of the cold wave front, the oxytrichas react immediately to the thermal stimulus, creep backwards at very high velocity along uninterrupted linear tracks, and thus move away from the cooling source. No specific behavioral response was ever observed in the static microgradient conditions. At 8.5 degrees C, despite their inertness, the ciliates are still able to behave adaptively, reacting immediately and orientatedly, once a directional factor (the cold-repelling thermal gradient) arises in an isotropic environment. This is similar to their behavior in the symmetric warm attracting thermal gradient.

The interface between air and water: a perturbation source eliciting adaptive behaviour in ciliates.

Interference with the water-air interface, both direct (by contact with a flat, rigid surface) and indirect (by inducing a meniscus) caused the ciliated protozoa we investigated to actively collect in the water column or on the substrate directly under the area of altered surface tension. A crowding effect is observed in this "rest area" reaching plateau values within one hour after onset of the experiment. The simple experimental procedures described here induced analogous behaviour in both Paramecium caudatum (a swimmer) and Oxytricha bifaria (a crawler). The ciliates seem in this reaction to be seeking a refuge from vibrations transmitted by the free interface. Our discovery is discussed in its implications for the adaptive biology and ecology of these micro-organisms.